1. Field of the Invention
The present invention relates to a method to enhance the efficacy of a vaccine by administration of a chemokine in conjunction with the vaccine. The present invention also relates to compositions useful in the practice of this method.
2. Description of Related Art
DNA immunization is presently being developed as an inexpensive and safe means for providing immunizations to large numbers of people. One of the shortcomings of this approach is the relatively weak immune response triggered by DNA vaccines. The present invention provides for the administration of select chemokines to enhance the immune response to DNA vaccines.
Because of their ability to attract discrete sub-populations of leukocytes to sites of inflammation and antigen presentation, chemokines are key agents in eliciting immune responses. In addition, chemokines reportedly can influence the T helper response towards a Th1 (i.e. cell mediated) or Th2 (i.e. humoral) profile, according to the differential distribution of receptors on target cells.
Preferred chemokines according to the practice of the present invention include RANTES (regulated on activation, normal T cell expressed and secreted), a chemokine which has receptors on both Th1 and Th2 cells, MCP-1 (monocyte chemoattractant protein), which is Th1-type associated, and the Th2-type associated chemokine BLC (B lymphocyte chemoattractant) and MDC (macrophage-derived chemoattractant). The inventors have studied the immuno-modulating properties of these chemokines in conjunction with DNA immunization, using HIV-1BaL gp120 and membrane-bound HIV-1BaL gp160 as antigens.
The inventors have discovered that chemokines modulate immune responses according to their Th-type polarization. Accordingly, the investigated chemokines can be ranked in the following order of induction of Th-2 vs Th1 responses: BLC (inducing mostly humoral responses), MDC, MCP-1 and RANTES (inducing mostly cellular responses). Quantitatively, MCP-1 was the strongest inducer of cellular responses, while the BLC induced the strongest humoral response.
These results are of great importance from the perspective of developing and optimizing vaccination regimens, in particular, against viral infections or cancer, but also, in general where directed or combined Th-type responses are sought. DNA Vaccines
DNA-based vaccines combine safety, ease of use, handling and modification and cost-effective production with the efficacy and effectiveness of live-attenuated vaccines. They are capable of eliciting both strong humoral and cell-mediated immunity (Moelling, K., 1997, Donnelly, J. J. et al., 1997; Montgomery, D. L. et al., 1997). Therefore DNA immunization represents a new approach for prevention (vaccination) and treatment (immune-based therapy) of infectious and neoplastic diseases. It has been shown that many factors contribute to the outcome of the immune response (Cohen, A. D. et al., 1998; Chun, S. et al., 1998) and might therefore be exploited. The present inventors have discovered that co-administration of chemokines with DNA vaccines significantly affects the immune response, both in amplitude and quality. The chemokines used in the empirical work presented herein are representative of classes of chemokines which attract specific subsets of lymphocytes and/or antigen presenting cells.
The surprising discovery that genes in plasmid expression vectors are expressed in vivo after intramuscular (i.m.) injection and that this expression stimulates an immune response against the plasmid-encoded proteins, has led to the concept of ‘DNA vaccination’ (Wolff et al., 1990). It has been shown that upon plasmid injection both antigen-specific antibody (Ab) responses and cytotoxic T Cell (CTL) responses are produced (Tang, D., 1992; Wang et al., 1993; Ulmer et al., 1993), without damaging the muscular tissue. The DNA vaccination procedure is safe because it uses only a part of the genome of the pathogen, hence making an active infection impossible. The ability to express virtually any antigen, or antigen combination by genetic engineering, coupled with efficacy and safety, has fueled the great popularity of DNA vaccines.
A considerable number of studies have focused on HIV and cancer vaccines (Fomsgaard, A., 1999; Barnett, S. W. et al., 1998; Kennedy, R. C., 1997; Oppenheim, J. et al., 1996; Burton, D. R. & Moore, J. P., 1998), and phase III studies using HIV-based vaccines have already begun (VaxGen (1999). However, current DNA vaccines still need improvement, since it is likely that both strong CTL responses and high neutralizing Ab titers are required, at least for some vaccines to be effective (Corel, et al., 1998), and indeed, most of the vaccines used have only limited efficacy in inducing efficient humoral responses (AIDS Alert, 1998).
DNA immunization is generally performed using immuno-stimulants (adjuvants) (Falo, L. D. Jr. & Storkus, W. J., 1998, Sasaki, S., 1998). Those adjuvants can be of very different nature (Allison, A., 1997; Gupter, R. K. and Siber, G. R., 1995; Cox, J. C. and Coulter, A. R., 1997): currently used preparations include bacterial cell-wall derivatives (Freund's adjuvant), oil-based emulsions (MF-51, SAF-1), aluminum salts (alum), saponine derivatives (QS21), or polymers (polyphosphazene). More recently cytokines and chemokines have been proposed as “natural” adjuvants, in both DNA-based and traditional immunization protocols (Xin, K. Q., 1999; Sin, J. I. et al 1999; Wang, B. et al., 1993; Okada, E. et al., 1997).
Polypeptide Vaccines
Classical protein vaccination protocols are effective in inducing high humoral response (Sha, Z. et al., 1999), unlike, for the time being, DNA immunization. The immunization with protein also allows the establishment of an accurate dose dependency, which is not possible using DNA vaccination protocols, since in DNA vaccination the ratio of administrated and finally expressed DNA remains unknown. On the contrary, the exact amount of injected protein is known and there is no delay via the process of transcription, translation and secretion, so that when chemokines are coadministered, their potential enhancing effect on antigen presentation will occur in hours rather than days.
Protein immunization is not very effective for inducing cell-mediated immunity, due to the route of antigen processing and presentation. However, depending on the pathogen, cellular responses may be crucial (Connor, R. I. et al., 1998). Special adjuvants have been designed to circumvent this hurdle (Sheikh, N. A. et al., 1999); however, these formulations using these adjuvants have the potential disadvantage of denaturing the protein, and thereby possibly preventing the elicitation of a relevant antibody response (VanCott, T. C. et al., 1997).
Another advantage of protein immunization lies in the ability to quickly study potential B or T cell epitopes. Peptide vaccination has been shown to be promising in both T cell induction and suppression (Ruiz, P. J., 1999: Toes, R. E., 1996), which has lead to extensive studies in pharmaceutical drug design, although T cell epitopes, in contrast to B cell epitopes, would be limited to the major MHC-haplotypes that present them.
Protein vaccination is as safe as DNA vaccination since there is no risk of infection by a live pathogen. In addition, autoimmune or tolerance reactions, which might be elicited by long-term expression of plasmid-based vaccines (Mor, G., 1997), are less likely to be induced by protein immunization. Therefore, protein-DNA mixed protocols have the potential of combining the advantages of DNA and protein immunization, with the lowest risk of inducing undesired or ineffective responses (Bruehl, P. et al., 1998; Richmond, J. F. L. et al., 1998; Barnett, S. W., 1998).
Chemokines
Chemokines are a family of small cytokines, that are released in response to infection together with other inflammatory cytokines (Mackay, C. R., 1997). Their molecular masses range from 6-14 kDa (Ward, S. G., 1998), and they all have related amino acid sequences which are between 20 and 50% sequentially homologous. Chemokines are multiple mediators, but were first studied as inducers of chemotaxis of specific leukocytes (Nelson, P. I. & Krensky, E. M., 1998; Kim, C. H. et al., 1999; Moser B, 1998). Further studies have revealed that chemokines also stimulate lymphocyte development, angiogenesis, degranulation of granulocytes, respiratory bursts and the release of lysosomal enzymes in monocytes. Furthermore, chemokines were shown to reduce the threshold of responsiveness of immune cells to other inflammatory mediators (Taub, D. D., 1996). These properties render chemokines particularly important in localizing and enhancing inflammation.
Chemokines are divided into four different subfamilies, according to the position of the first two cysteines in their primary sequence; the α-chemokine subclass bears a CXC-motif, where the two cysteines (C) are separated by one amino acid; the β-chemokines contain a CC motif, the γ-subclass lacks one cysteine residue, and is yet represented only by one member, lymphotactin, and in δ-chemokines, or CX3C subclass, the two cysteines are separated by three amino-acids. These cysteine residues form disulfide bridges with two other cysteines located further downstream in the primary sequence, thus stabilizing the tertiary structure of these chemokines.
Both primary and tertiary structures are crucial in inducing differential chemoattractant responses, determining binding to specific receptors (Baggiolini, M., 1997). However, the chemokine/chemokine receptor system is in part redundant, since overlapping ligand specificity by interaction with several chemokine receptors has been shown (Campbell, J. J. et al., 1997). This relatively complicated network of ligand/receptor interaction has been tentatively explained by the “multistep navigation model” (Foxman, F. F., 1997), according to which migrating lymphocytes follow a hierarchy, of chemotactic gradients. Leukocytes will encounter and respond to multiple chemoattractant signals in a complex spatial and temporal pattern. Those signals guide the leukocytes from the vascular system to the site of inflammation by inducing sequentially their rolling through the endothelium and the firm adhesion and extravasation at the peak of the gradient, namely, where most adhesion molecules are expressed. Additional leukocytes may be directed via other signals through the tissue to the site of inflammation. During this migration, several receptors may be triggered simultaneously and/or successively on both leukocytes as on the cells involved in their ‘guidance.’ This model therefore proposes specific blockage sites for therapeutic purposes, such as treating allergic and autoimmune diseases, with fewer side effects than common immunosuppressants. Specific blocking of chemokine-receptors could be achieved by specific antagonists, such as monoclonal Abs or modified chemokine derivates (Simmons, G. et al., 1997; Chen, J. D., 1997; Baggliolini, M. and Moser, B., 1997; Wu, L., 1997).
Chemokine receptors are designated CXCR followed by a number when binding cc-chemokines and CCR followed by a number when binding β-chemokines. All chemokine receptors belong to the group of G-protein-coupled seven transmembrane domain receptors (Baggiolini M, 1997).
HIV Vaccines
Human immunodeficiency virus (HIV) induces a persistent and progressive infection leading, in the vast majority of cases, to the development of the acquired immunodeficiency syndrome (AIDS) (Barre-Sinoussi et al., 1983, Science 220:868-870; Gallo et al., 1984, Science 224:500-503). The HIV envelope surface glycoproteins are synthesized as a single 160 kilodalton precursor protein, which is cleaved by a cellular protease during viral budding into two glycoproteins, gp41 and gp120. gp41 is a transmembrane glycoprotein and gp120 is an extracellular glycoprotein which remains non-covalently associated with gp41, possibly in a trimeric or multimeric form (Hammerskjold, M. and Rekosh, D., 1989, Biochem. Biophys. Acta 989:269-280). The V3 loop of gp120 is the major determinant of sensitivity to chemokine inhibition of infection or replication (Cocchi et al., 1996, Nature Medicine 2:1244-1247; and Oravecz et al., 1996, J. Immunol. 157:1329-1332).
Although considerable effort is being directed towards the design of effective therapeutics, currently no completely curative anti-retroviral drugs against AIDS exist. The HIV-1 envelope proteins (gp160, gp120, gp41) have been shown to be the major antigens for neutralizing anti-HIV antibodies present in AIDS patients (Barin et al., 1985, Science 228:1094-1096). These proteins are promising antigen candidates for anti-HIV vaccines. Several groups have begun to use various portions of gp160, gp120, and/or gp41 as immunogenic targets for the host immune system (see, for example, Ivanoff, et al., U.S. Pat. No. 5,141,867; Saith, et al., PCT publication WO 92/22654; Shafferman, A., PCT Publication WO 91/09872; Formoso, et al., PCT Publication WO 90/07119). Therefore, methods to increase the efficacy of vaccines against HIV, especially vaccines using gp120 as the antigen, are needed.
Additionally, a novel vaccine technology, designated genetic vaccination, nucleic acid vaccination or DNA vaccination, has been explored to induce immune responses in vivo. Injection of cDNA expression cassettes results in in vivo expression of the encoded proteins (Dubensky, et al., 1984, Proc. Natl. Acad. Sci. USA 81:7529-7533; Raz, et al., 1993, Proc. Natl. Acad. Sci. USA 90:4523; Wolff, et al., 1990, Science 247:1465-1468), with the concomitant development of specific cellular and humoral immune responses directed against the encoded antigen(s) (Wang, et al., 1995, Hum. Gene Ther. 6:407-418; Ulmer, et al., 1993, Science 259:1745-1749; Tang, et al., 1992, Nature 356:152-154; Michel, et al., 1995, Proc. Natl. Acad. Sci. USA 92:5307-5311; and Lowrie, et al., 1994, Vaccine 12:1537-1540). Humoral and cellular responses have been induced to HIV-1 and SIV antigens through various applications of this technology in macaques (Wang, et al., 1995, Virology 221:102-112; Wang, et al., 1993, Proc. Natl. Acad. Sci. USA 90:4156-4160; and Boyer, et al., 1996, J. Med. Primatol. 25:242-250) as well as mice (Wang, et al., 1995, Virology 221:102-112; Lu, et al., 1995, Virology 209:147-154; Haynes, et al., 1994, AIDS Res. Hum. Retroviruses 10 (Suppl. 2):S43-S45; Okuda, et al., 1995, AIDS Res. Hum. Retroviruses 11:933-943).
Recently, Lekutis, et al. (1997, J. Immunol. 158:4471-4477), assessed the TH cell response elicited by an HIV-1 gp120 DNA vaccine in rhesus monkeys by isolation of gp120-specific, MHC class II-restricted CD4+T cell lines from the vaccinated animals. Lekutis, et al. showed that the isolated cell lines proliferated in response to APC in the presence of recombinant gp120, as well as to APC expressing HIV encoded env protein. Lekutis, et al. further showed that these cell lines responded to env by secreting IFNΓ and IFNα without appreciable IL-4 production. These results demonstrate that the animals exhibited a cellular immune response to the DNA vaccine. Boyer, et al. (1997, Nature Medicine 3:625-532), inoculated chimpanzees with an HIV-1 DNA vaccine encoding Env, Rev, and Gag/Pol, and found that the immunized animals developed specific cellular and humoral immune responses to these proteins. After challenging the immunized animals with a heterologous chimpanzee titered stock of HIV-1 SF2, Boyer, et al. further found, using a Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) assay, that those animals vaccinated with the DNA vaccine were protected against infection whereas the control animals were not so protected.
Kim, et al., (1997 J. Immunol. 58:816-826), investigated the role of co-delivery of genes for IL-12 and GM-CSF along with DNA vaccine formulation for HIV-1 antigens env and gag/pol in mice. Kim, et al. observed a dramatic increase in specific CTL response from the mice immunized with the HIV-1 DNA vaccine and IL-12. Kim et al. also observed that the co-delivery of IL-12 genes resulted in the reduction of specific antibody response, whereas the codelivery of GM-CSF genes resulted in the enhancement of specific antibody response. Kim, et al. further observed that co-delivery of IL-12 gene with a HIV DNA vaccine results in splenomegaly (Kim, et al. 1997, J. Immunol., 158:816-826), which has been shown in mice to have toxic effects such as weight reduction or even death (Eng, et al., 1995, J. Exp. Med. 181:1893; Stevensen, et al., 1995, J. Immunol. 155:2545; and Orange, et al., 1995, J. Exp. Med. 181:901).
Notwithstanding the recent developments in the field of HIV DNA vaccines, there still exists a need for a method to enhance the efficacy of a vaccine, especially an HIV DNA vaccine. For instance, both cellular and humoral immune responses are needed to control an HIV-1 infection (Boyer, et al., 1997, Nature Medicine 3:625-532). The induction of both cellular and humoral immune response by the Berjer, et al. method is still quite low because only one of the three immunized chimpanzees developed both cellular and humoral responses. Similarly, although co-delivery of an IL-12 encoding gene with a HIV DNA vaccine, as described in Kim, et al. (1997, J. Immun. 158:816-826), may have enhanced the cellular immune response, this co-delivery also decreased the humoral response.
Citation of any of the references discussed hereinabove shall not be construed as an admission that such reference is prior art to the present invention.